Introduction
The life cycle of most teleost fishes embraces a crucial phase known as larval period, during which larvae undergo many evolutionary events (e.g., start of the exogenous feeding, the yolk-sac reduction and the inflation of the swim bladder) that chiefly affect their morphological, behavioral and physiological status (Urho, 2002). Those changes appear to have a different impact on the performance (often referred to as quality) of fish larvae (Kjørsvik et al., 2003). Among others, growth-related traits have been commonly used as parameters to assess larval quality and were reported to significantly vary between the specimens even of the same cohort (Mun et al., 2019; Wang et al., 2021). Despite that, mechanisms and factors leading to different performances during the early life history of fish are largely unknown. Therefore, using a family-based experimental approach, we intended to thoroughly investigate the relation between zootechnical and transcriptomic profile of Eurasian perch (Perca fluviatilis) larvae in order to gain deeper insight into the developmental processes influencing larval quality.
Material and methods
Sixteen unique families of Eurasian perch were created by controlled reproduction. Eggs were fertilized in vitro using cryopreserved semen (Judycka et al., 2022), and then incubated in a recirculating aquaculture system (RAS). The hatched larvae were reared for 30 days following the standardized procedure described by Palińska-Żarska et al. (2020) and during the larvicultural period several zootechnical data were collected (e.i., length, weight, swim bladder inflation rate, cannibalism rate, mortality). In addition, larvae at mouth opening stage (right after hatching) were sampled for transcriptomic analysis. Obtained RNA-sequencing data were mapped to the reference genome P. fluviatilis and later processed with Weighted Gene Coexpression Network Analysis (WGCNA) using a R package (Langfelder & Horvath, 2008). Gene ontology (GO) enrichment analysis was carried out using ShinyGO (Ge et al., 2020).
Results
The analysis revealed statistically significant differences (p < 0.05) between families for length and weight at hatching and throughout the rearing period (first feeding, oil droplet reduction, weaning and at the end of the larval period), cannibalism rate, mortality and specific growth rate (SGR). There was no significant difference for the swim bladder inflation effectiveness (SBIE).
WGCNA of RNA-seq data showed interactions between genes (grouped in different modules according to their Pearson correlation) and the zootechnical data collected. There were significant modules related to weight at mouth opening, cannibalism and mortality but mostly to the length at mouth opening. Specifically 5 modules were positively correlated to length at mouth opening and 3 modules were negatively associated to it. We found that in positively length-related modules, ribosome biogenesis, translation processes, rRNA processing were most enriched biological processes. On the contrary, in modules negatively correlated to length, the most enriched processes included neurogenesis, neuron projection development, cell-cell signalling, cell projection and cytoskeleton organization.
Discussion
Here, for the first time, we attempted to explore differences in larval performances by comparing 16 different families in Eurasian perch. WGCNA highlighted the importance of length data at hatching and the results indicate candidate genes and biological processes providing ground basis for understanding mechanisms leading to length heterogeneity in fish.
Ribosomal proteins (RP), such as rps19 and rps6, being positively correlated with the length of newly hatched larvae, are suggested as potential biomarkers in controlling larval development. Furthermore, the analysis outlines a negative link between genes related to cytoskeleton, nervous system and the length trait. During the early life stages, larvae are still evolving and genes involved in the formation of the cytoskeleton and axon growth could play a crucial role in larval performance. In this context, IQ motif containing GTPase activating protein 2 (iqgap 2) gene could largely influence the early developmental stages of larvae (Fang et al., 2015).
The outcomes of our study provide new insights into the processes involved in the determination of larval quality and propose WGCNA as a robust approach to investigate the transcriptomic profile of Eurasian perch larvae.
Acknowledgment
This work was funded by National Science Center of Poland (SONATA BIS project, number UMO-2020/38/E/NZ9/00394).
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